Glucocorticoid receptors in vivo are phosphorylated in the absence of hormone and become hyperphosphorylated in the presence of glucocorticoid agonist but not antagonists (Ortí, E., Mendel, D.B., Smith, L.I., and Munck, A. (1989) J. Biol. Chem. 264, 9728-9731). As a preliminary step to elucidating the functional significance of receptor phosphorylation, we have identified seven phosphorylated sites on the mouse receptor. Tryptic phosphopeptides from 32P-labeled receptors were purified from glucocorticoid-treated mouse thymoma cells (WEHI-7) and from stably transfected Chinese hamster ovary cells (WCL2) that express large numbers of mouse receptors. Phosphopeptide maps of receptors from these two cell types were almost indistinguishable. Solid phase sequencing revealed phosphorylation at serines 122, 150, 212, 220, 234, and 315 and threonine 159. Serines 122, 150, 212, 220, and 234 and the sequences surrounding them are conserved in the homologous regions of the rat and human receptors, but threonine 159 and serine 315 have no homologues in the human receptor. The seven phosphorylated sites are in the amino-terminal domain of the receptor. All but serine 315 are within transactivation domains identified in the human and/or rat receptors. Serines 212, 220, and 234 are in a highly acidic region that in the mouse receptor is necessary for full transcription initiation activity and reduces nonspecific DNA binding. Serines 212, 220, and 234 and threonine 159 are in consensus sequences for proline-directed kinase and/or p34cdc2 kinase. Serine 122 is in a consensus sequence for casein kinase II whereas serines 150 and 315 do not appear to be in any known kinase consensus sequence. The location of many of these sites suggests a role of phosphorylation in transactivation.